Running a DNA Gel
FOR LABELING GELS, PLEASE SEE THE LABELING GELS ARTICLE. Follow the guidelines in this article for all gel labeling. Pouring the gel Make your gel first. Dissolve the required amount of the cheap stuff agarose (chemical shelf) (P/N: Fisher, BP160-500) into 1X buffer (TAE). Note: there is often a backup stock of agarose in the stockroom if you don't see any on the chemical shelf- we go through it quickly. Melted agarose is VERY hot and VERY slippery. Treat it accordingly if you manage to boil it over- try to never fill a container more than 1/3 full with it when boiling. 1. Set up your gel pouring equipment. The trays and individual gel forms are in various drying trays near sinks throughout the lab. Take from there and return to there when finished. Also grab the bar that forms the wells for the gels, appropriate to whatever size tray your using to pour these. 2. Insert gel forms into the tray, mount the well forming bar, and use a closed, inverted microfuge tube to create a little extra space in the wells by inserting just the edge of the tube under the well forming bar at the ends on the side of the tray. 3. Take your gel, which is probably in solid form in a jar (shelf above Mark's bench?) and go to the other lab room to nuke it in the microwave. Start with one minute, give it a little shake, do another minute, more shake, then maybe one more minute at most. then grab the Ethidium Bromide (P/N: Sigma, E1510-10ML) off the student helper's bench and return to your lab bench. Note: Ethidium Bromide is light sensitive, and YOU are Ethidium Bromide sensitive. Wear nitrile gloves, do not get it on or in you anywhere. If you've never worked with EtBr before, please have another lab member walk you through proper precautions. 4. Pour your gels individually straight from the jar into the tray, up to approximately the level of the lower side of each gel form (ie, dont overflow). After each gel is poured, use a P2 pipettor and the tiny pipette tips and spread (2 uL in large gels, 1 uL in small) EtBr throughout your gel. Try to avoid swirling the hot gel so much that if forms bubbles, but if you do, use your tip to move them off to the side and out of the way. Running the Gel There is an alternate way of running the gel, which works just as fine as this protocol, but involves staining after electrophoresis. Here is the link to that page. Once you have your poured gel and it has solidified, get your PCR'd samples or other DNA samples ready to run on the gel: 1. Add 5uL of 6X loading dye to each PCR test well if the samples were run as 25uL total in the PCR wells. 2. Create a ladder to compare band sizes to. We have been using a 1kb ladder; several options are available from Biochem Stores. These will be on the outsides of your gel, in the end wells. If you need as many wells as possible, you can choose to run only one ladder, but having one on each end is helpful if you have the space. The ladder is only 1uL in the gel wells, and each only gets 1/6uL of loading dye, so if you're going to use two ladders thats 2uL of ladder and 0.33uL of dye in an extra PCR well. 3. Make a diagram of your gel in your notebook with labels reading from left to right that describe which samples are in which gel well. Stick the ladders on the ends. 4. Load the gel by using the tiny (10uL) pipette tips and an appropriate pipettor. The ladders need 1uL of your mix of ladder+dye, and each test sample should be run at 10uL (P20 pipette). When loading the gel wells, it is helpful to brace the pipette against your off hand, and brace that hand against the table or the edge of the electrophoresis kit. If you are unsure of whether or not your tip is in the well, gently wiggle your tip and see if the gel moves around with it slightly, if so, you are in the well. Carefully depress all of the sample out of the tip and try not to drag any out the top of the well with your tip when you remove it. 5. When your gel is loaded, grab your timer, put the cover on the gel kit. Some kits have timers, for these you set the time and the voltage (100 volts), then press ON. Other kits you first have to press ON, then set the voltage, and these do not have timers. Regardless of whether or not your kits has a timer, set your own timer. 6. Wait 30 minutes... P.S. 6 X DNA loading Dye: # 3.75ml glycerol (30%) # 25mg bromophenol blue (0.25%) # dH2O to 10mL Taking a Picture 1. Take a picture using the Gel Camera in the Greenberg lab on the third floor, 4-352. Your uname and pwd to login to this windows box are your Buck email user name and pwd. AGAROSE: P/N: Fisher, BP160-500 ETHIDIUM BROMIDE: P/N: Sigma, E1510-10ML ADD DETAILS ON SETTINGS AND WHAT TO SAVE HERE